Bone growth in children occurs by endochondral ossification in cartilaginous growth plates at the ends of long bones. In the last decade, analysis of transgenic constructs has become the single most powerful approach for the study of endocrine, paracrine and autocrine regulators acting during the chondrocytic differentiation cascade. However, despite major advances in delineating the cellular and molecular events that define skeletal growth and its regulatory circuitry, relatively little is known about the physiology of the vascular access that endocrine (and potentially paracrine) regulators require to reach growth plate chondrocytes, and how these molecules move within the spatially heterogeneous growth plate matrix. To a large extent this reflects a lack of methods to study these questions under real time conditions in living animals. This is particularly challenging for studying the growth plate, which has three conceptually different blood supplies: epiphyseal, metaphyseal, and perichondrial (including a ring vessel and a plexus.) We will apply a novel approach to these questions by utilizing multiphoton microscopy (MPM) for multi-hour in vivo imaging of the murine proximal tibial growth plate. Our experimental system is anesthetized 4-5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter (Col II/GFP). The specific aims are summarized as: To test the hypothesis that the rate of delivery into the growth plate varies for molecules of different size, by directly imaging the arrival of IV injected fluorescent tracers (starting with dextrans but moving to known biologically significant regulators of chondrocytic differentiation.) To test the hypothesis that tracers of different molecular weight reach the growth plate through different vascular routes and travel within the growth plate in different matrix subcompartments. For the epiphyseal vasculature the analysis will include time points prior to full development of the secondary center of ossification. To image cellular turnover events at the metaphyseal chondroosseous junction in real time, as an "internal clock" for correlation with data on rates of bone elongation. These analyses are relevant to the understanding of growth aberrations such as trauma with premature closure and disruption of the periosteum leading to overgrowth. Detailed functional knowledge of vascular routes to the growth plate, and movement of molecules within the growth plate, is essential as thought is given to targeting therapeutic agents to the growth plate.